1. Field of the Invention
The present invention relates generally to the field of protein biochemistry. More particularly, it concerns improved methods for the renaturation and refolding of polypeptides aggregates.
2. Description of Related Art
The aggregation of proteins is of significant concern in the biotechnology, pharmaceutical and medical communities. In vitro, aggregation is observed in virtually every step in the production, refolding, purification, storage and shipping of protein therapeutics (Carpenter et al., 1997; Clark et al., 1999). In vivo, numerous pathogenic conditions in humans (e.g., Alzheimer's disease, Parkinson's disease and prion diseases (Kelly, 1996; Kelly, 1998; Prusiner, 1998; Scherzinger et al., 1999)) have protein aggregation and formation of insoluble deposits associated with the condition and, as a result, research on the characterization of the aggregates and mechanisms of aggregation in these diseases is an active area of medical research.
Aggregation in human protein deposition diseases, which displays organization in the form of insoluble fibrils, has brought increased significance to the study of protein misassembly and aggregation processes in general (Wetzel, 1999). Investigation into the reversal of aggregation and precipitation processes has immediate practical implications for the production, purification and delivery of therapeutic proteins.
In the production of therapeutic proteins, aggregated precipitates (e.g., inclusion bodies) is commonly reversed by dissolution of the precipitated aggregates in the presence of high concentrations of chaotrope (e.g., 6 M guanidine hydrochloride). Such harsh conditions result both in disaggregation (solubilization) and in nearly complete unfolding of the protein. Commonly, refolding is effected by removal of the chaotrope via dialysis or dilution to protein concentrations of ca. 10 to 50 μg/mL (Clark et al., 1999). Because refolding is commonly a first-order (in protein concentration) process and aggregation a second-order or higher process, refolding yields are improved at lower protein concentrations (Clark et al., 1999). Soluble aggregates are often separated from the native protein by costly and time consuming column chromatographies. Separated soluble aggregates are typically discarded, thus reducing overall protein yields and substantially increasing protein production costs. An alternative to chaotropic dissolution to dissolve insoluble aggregates or column purification to remove soluble aggregates is disaggregation by pressure (Foguel et al., 1999; Gorovits & Horowitz 1998; St. John et al., 1999).
Several research groups have exploited the ability of pressure to dissociate native protein oligomers (Silva & Weber 1993). In addition, others have explored the use of pressure to disaggregate and refold proteins from soluble non-native protein aggregates (Foguel et al., 1999; Gorovits & Horowitz, 1998) and precipitated, insoluble non-native aggregates (St. John, et al., 1999). Gorovits and Horowitz 1998 used high pressure to inhibit formation of soluble aggregates in 3.9 M urea solutions of rhodanase, and to reverse the formation of soluble aggregates. However, Gorovits & Horwitz (1998) report that “pressure . . . is not able to reverse large aggregates.” Treatment at 2.4 kbar for 90 minutes of soluble aggregates formed from P22 tailspike protein reduced aggregate levels from 41.1 to 17.6% (Foguel et al., 1999).
St. John et al. (1999) used high pressure to dissolve and recover native protein from large, insoluble aggregates at pressures on the order of 200 MPa, including aggregates formed as inclusion bodies. High yields at high protein concentrations of refolded, active human growth hormone, lysozyme and β-lactamase from insoluble, precipitated aggregates were achieved using non-denaturing concentrations of guanidine hydrochloride in combination with pressure or pressure in the absence of guanidine hydrochloride (St. John et al., 1999). In the specific case of insoluble aggregates of lysozyme containing non-native intermolecular covalent disulfide bonds that served to crosslink the insoluble precipitates, redox shuffling agents such as mixtures of reduced and oxidized glutathione were used in combination with high pressure and 0.8M guanidine HCl to afford high yields of folded, biologically active protein. In the specific case of aggregated and precipitated and aggregated human growth hormone, low levels of a chaotrope such as guanidine HCl were used to optimize recovery of soluble, native protein. In the specific case of B-lactamase inclusion bodies, addition of guanidine HCl did not increase yield of biologically active B-lactamase, but did result in higher solubilization of contaminating proteins.
Nonetheless, improved methods for the high-pressure dissociation of protein aggregates and refolding of solubilized proteins are desired.